Mouse anti gapdh 6c5

The 100,000 × g pellets from N2a NM-HA^sol and N2a NM-HA^agg clones s2E and 1C (adjusted to similar amounts of NM-HA protein, 1:2 and 1:4 dilutions) were resuspended in 2% SDS and loaded onto a preequilibrated nitrocellulose membrane (0.2-µm pore size; Invitrogen) with a Whatman Minifold I dot blotting apparatus (GE Healthcare). Wells were rinsed five times with 200 µl of filter trap assay buffer (1% SDS and 50 mM EDTA in PBS). The membrane was incubated overnight at 4°C with anti-HA 3F10 antibody (1:1,000; Roche) after blocking in 5% skimmed milk for 1 h. For Western blot analysis, protein concentrations were measured by Quick Start Bradford protein assay (Bio-Rad) and proteins were separated on NuPAGENovex 4 to 12% bis-Tris protein gels (Life Technologies) and then transferred onto a polyvinylidene difluoride membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed with mouse anti-Alix (1:1,000; BD Bioscience), rabbit anti-flotillin rbt 3253 (1:1,000; NEB), rabbit anti-Tsg101 ab30871 (1:500; Abcam), rat anti-HA 3F10 (1:1,000; Roche), rabbit anti-calnexin C4731 (1:1,000; Sigma), mouse anti-GAPDH 6C5 (1:5,000; Abcam), and mouse anti-Hsc/Hsp70 N27F3-4 (1:1,000; ENZO) antibodies. The membrane was incubated with Pierce ECL Western blotting substrate (ThermoFisher Scientific) according to the manufacturer’s recommendations.

For Western blot analysis, protein concentrations were measured by Quick Start^TM Bradford Protein assay (Bio-Rad) using the plate reader Fluostar Omega BMG (BMG Labtech) and the corresponding MARS Data Analysis Software (BMG Labtech). Proteins were separated on NuPAGE®Novex® 4–12 % Bis-Tris Protein Gels (Life Technologies) followed by transfer onto a PVDF membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed using mouse anti-LDL receptor (1:500; NovusBiologicals); mouse anti-Alix (1:1000; BD Bioscience); rat anti-HA 3F10 (1:1000; Roche); mouse anti-GAPDH 6C5 (1:5000; Abcam); mouse anti-Hsc/Hsp70 N27F3-4 (1:1000; ENZO); mouse anti-VSV-G 5D4 (1:1000; Sigma); rat anti Sup35 M domain (1:10; hybridoma supernatant);^{88 (link)} rabbit anti-Tau ab64193 (1:1000; Abcam); rabbit anti-Flotillin 1 ab133497 (1:1000; Abcam); mouse anti-SARS-CoV-2-spike S GTX632604 (1:1000; GeneTex); rabbit anti-hACE2 ab15348 (1:1000; Abcam); rabbit anti-clathrin heavy chain (1:1000; Abcam); rabbit anti-GFP (1:5000; Abcam). The membrane was incubated with Pierce^TM ECL Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer´s recommendations and imaged with the Imaging system Fusion FX (Vilber Lourmat).

The PCa cell lysates (DU145, LNCaP,
and PC3) were prepared using the RIPA buffer. The lysates were briefly
sonicated and centrifuged, and the protein concentrations were determined
using the BCA protein assay kit (Thermo Fisher, CA, USA). A total
of 40 μg of cell lysates was separated using 10% SDS-polyacrylamide
gel electrophoresis and transferred on to Amersham Hybond ECL nitrocellulose
membranes (Amersham, QC, Canada). The membranes were blocked using
5% skim milk in phosphate-buffered saline with 0.1% Tween-20 for 1
h and then incubated with specific primary antibodies overnight at
4 °C followed by incubation with HRP-conjugated secondary antibodies
for 1 h at room temperature. Specific protein bands were visualized
using an ECL detection kit (Amersham). The primary and secondary antibodies
used were rabbit anti-ALDH1A1 (D9J7R, Cell signaling Technology, UK),
mouse anti-GAPDH (6C₅, Abcam, Cambridge, UK), rabbit anti-ALDH1A3
(N₂C2, GeneTex, California, USA), mouse anti-ALDH3A1 (G-2,
Santa Cruz Biotechnology, CA), anti-rabbit HRP (Dako), and goat anti-mouse
HRP (Abcam, Cambridge, UK).